Expensive equipment, highly-trained personnel, and the need for a clinical laboratory setting precludes routine nucleic acid testing (NAT) for infectious disease in most of the developing world and even in many resource-limited parts of the United States, leading to wide disparities in health care worldwide. A fast, sensitive, low cost but a facile NAT method for robust detection of specific agents at the point of care (POC) would help bring molecular diagnostics to everyone. The goal of this application is to demonstrate feasibility of a complete field-appropriate molecular detection system suitable for use in low resource settings for the detection of three important arboviruses, Chikungunya (CHIKV), Zika (ZIKV) and dengue (DENV). The innovative technology that is the basis of this application is a new thermostable polymerase, OmniAmp, which has innate reverse transcriptase (RT) activity. OmniAmp is suitable for application in a promising NAT alternative to the polymerase chain reaction (PCR) called loop mediated isothermal amplification (LAMP). Since LAMP is isothermal, it does not require specialized instrumentation plus it is much faster than PCR. LAMP is also resistant to inhibitors in crude sample preparations. The proposed assay will be based on LAMP using OmniAmp polymerase, and performed on a simple, easy to use automated molecular detection (MDx) platform. Total assay time will be 40 minutes with minimal hands-on time and without need of any additional equipment, such as pipettes, centrifuge etc. Results will be displayed on-screen as positive or negative for a specific pathogen, minimizing error?s caused by user interpretation LAMP assays for detection of all major lineages of CHIKV and ZIKV, including strains now circulating in the Americas, and all 5 serotypes of DENV will be developed. Performance of LAMP assays will be compared to that of reference real time RT-PCR methods. For field use, a simple and rapid sample preparation method for extraction of nucleic acid from samples (whole blood and serum) will also be developed. In order to increase the shelf life of reagents, amplification reagents will be dried down. Analytical sensitivity and specificity will be evaluated by testing whole blood and serum samples by spiking them with different titers of CHIKV, ZIKV and DENV serotype, both individually and co-infection. By combining Lucigen?s capacity in amplification and detection with the expertise in arboviral research and fundamental capability provided by the University of Texas Medical Branch, a molecular detection system will be developed for detection and differentiation of CHKV, ZIKV and DENV, that can be implemented almost anywhere, worldwide. Successful completion of this project will lead to: ? Development of molecular diagnostic assay for detection and differentiation of three important arboviruses, CHIKV, ZIKV, and DENV at POC. ? Total assay time of 40 minutes including sample preparation and run time. ? Detection of multiple targets in a single run from single sample prep. ? Dried reagents for storage at ambient temperature. ? A technology platform that can be adapted for the detection of other pathogens.